This grant examines the participation of the DHPR beta1a subunit in excitation-contraction coupling in skeletal muscle. The central hypothesis is that protein-protein interactions between the DHPR beta1a subunit and RyR1 are essential for signals transmitted from the DHPR to the RyR1 channel during EC coupling. The hypothesis is tested by a combination of in-vitro recombinant protein and cell-based approaches. Proximity determinations using FRET are used to investigate a structural model proposed for beta1a. Recombinant protein and in-vitro expression approaches are used to map domains and molecular motifs in beta1a and RyR1 that bind to each other. Electrophysiological approaches implemented in primary cultured myotubes from beta1 KO and RyR1 KO mice investigate the functional consequences of the interaction of beta1a and RyR1. Aim 1 will develop a model of the domain organization of beta1a based on its homology to the MAGUK protein PSD-95. We propose to investigate the domain organization of beta1a using FRET in beta1a variants with fused CFP as the donor and covalently-bound FIAsH as the acceptor. Aim 2 will examine the role of the C-terminal domain of beta1a in EC coupling. Cell-based mutagenesis and in-vitro pull-down strategies are used to assess the functional significance of C- and N-terminal heptad repeats present in beta1a. Aim 3 will characterize a critical region of RyR1 that binds beta1a and determine functional correlates of the beta1a-RyR1 interaction. Cell-based mutagenesis and in-vitro pull-down strategies will determine the significance of a putative binding site for beta1a located in the 3490-3523 region of RyR1. A beta1a/RyR1 could bring about strong docking of the DHPR and RyR1, and/or could be essential to the generation of the signal that opens RyR1. These two functions, although seemingly dissimilar, may represent two manifestations of the same molecular interaction. The purpose of this application is to gain a detailed molecular insight on how DHPR beta1a interacts with RyR1, and to pursue the functional consequences of this interaction. This information is crucial for understanding the molecular basis of normal EC coupling as well as the molecular basis of diseased states in which EC coupling is drastically altered.